antimicrobial potential of extracts from stevia rebaudiana leaves

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ANTIMICROBIAL POTENTIAL OF EXTRACTS FROM
STEVIA REBAUDIANA LEAVES AGAINST BACTERIA
OF IMPORTANCE IN DENTAL CARIES
Fredy Gamboa1, Margarita Chaves2
1
Departament of Microbiology (School of Sciences) and Dental Research Center (School of Dentistry).
Dental Research Center (School of Dentistry). Pontificia Universidad Javeriana. Bogotá, Colombia.
2
ABSTRACT
In recent years, the antimicrobial activity of Stevia rebaudiana
Bertoni leaf extracts against a large number of microorganisms has been evaluated, but not its activity against
microorganisms of importance in dental caries. The aim of this
study was to evaluate the antibacterial activity of Stevia rebaudiana Bertoni leaf extracts against cariogenic bacteria.
Extracts were obtained from the dried Stevia rebaudiana
Bertoni leaves in hexane, methanol, ethanol, ethyl acetate and
chloroform. The antimicrobial activity of the 5 extracts against
16 bacterial strains of the genera Streptococcus (n= 12) and
Lactobacillus (n= 4) was evaluated by the well diffusion
method. Minimal inhibitory concentrations (MIC) of the
extracts in hexane, methanol, ethanol, ethyl acetate and chloroform on the 16 bacterial strains were respectively 30 mg/ml,
120 mg/ml, 120 mg/ml, 60 mg/ml and 60 mg/ml. The zones of
inhibition present at the MIC were variable, ranging from 9
mm to 17.3 mm. Our results suggest that inhibition zones with
a hexane extract are similar to those obtained with ethanol and
methanol, but the minimal inhibitory concentration (30 mg/ml)
is lower. For the four Lactobacillus species, the inhibition zones
obtained between 12.3 and 17.3 mm were somewhat larger with
ethyl acetate and chloroform extracts, suggesting they were the
most susceptible microorganisms.
Keywords: antiInfective agents - stevia - dental caries
POTENCIAL ANTIMICROBIANO DE EXTRACTOS OBTENIDOS
DE HOJAS DE STEVIA REBAUDIANA BERTONI SOBRE
BACTERIAS DE IMPORTANCIA EN CARIES DENTAL
RESUMEN
En los últimos años se ha evaluado la actividad antimicrobiana
de extractos obtenidos de hojas de Stevia rebaudiana Bertoni
sobre un gran numero de microorganismos. Sin embargo, no
existen evaluaciones en microorganismos de importancia en
caries dental. El objetivo de este estudio fue evaluar la actividad antibacteriana de extractos de hojas de Stevia rebaudiana
Bertoni sobre microorganismos cariogénicos. A partir de hojas
secas de Stevia rebaudiana Bertoni convertidas en polvillo se
obtuvieron los extractos en dichos solventes. La evaluación de
la actividad antimicrobiana de los 5 extractos sobre las 16
cepas bacterianas de los géneros Streptococcus (n=12) y Lactobacillus (n=4) se realizó por el método de difusión en pozo.
Las concentraciones mínimas inhibitorias (CMI) de los extrac-
tos de hexano, metanol, etanol, acetato de etilo y cloroformo,
sobre las 16 cepas bacterianas fueron respectivamente de 30
mg/ml, 120 mg/ml, 120 mg/ml, 60 mg/ml y 60 mg/ml, respectivamente. Los halos de inhibición determinados a las CMI
fueron variables, el de menor valor fue 9 mm y el de mayor fue
de 17.3 mm. Nuestros resultados, sugieren que los halos de
inhibición en el extracto de hexano son semejantes a los
obtenidos para el etanol y metanol, sin embargo, la CMI (30
mg/ml) es menor. En las 4 especies de Lactobacillus los halos
de inhibición obtenidos entre 13 y 17.3 mm, son ligeramente
mayores en los extractos de acetato de etilo y cloroformo,
sugiriendo que fueron los microorganismos más susceptibles.
INTRODUCTION
In physiological conditions, most bacteria in the
oral cavity are compatible with health. However,
under certain circumstances of the oral environment
and the condition and behavior of the host, these
bacteria may reveal their potential virulence and
cause disease1. Dental caries is considered to be an
alteration of the oral ecosystem with predominance
of pathogenic flora. The main microorganisms associated to caries production are, in order of fre-
quency: 1) Streptococcus mutans (mainly serotype
c) and to a lesser extent S. sobrinus and S. gordonii
and 2) Lactobacillus and Actinomyces species1,2.
Their participation in the dental caries generating
process has led to the development and implementation of measures for prevention and/or control1,3.
Among other strategies implemented to eliminate
and/or control the microorganisms involved in dental caries, chemical and antimicrobial substances
have been used. Antimicrobial biomolecules of
Vol. 25 Nº 2 / 2012 / 171-175
Palabras claves: agentes antiInfecciosos - stevia - caries dental
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F. Gamboa, M. Chaves
natural origin are also currently being explored for
use in adjuvant therapy4.
Plants have been widely used around the world as
traditional remedies in the treatment of diseases. It
is estimated that 66% to 75% of the world population currently uses plant-based medicines5. The main
aim of research into medicinal plants is to identify
plants that possess pharmacological activity, and
thus discover new substances or molecules having
antimicrobial activity that could be transformed into
medications by different chemical processes and
used to control or prevent infectious diseases6.
Research by Katsura et al.7 reports the bactericidal
activity of bakuchiol against S. mutans, S. sanguis,
S. salivarius, S. sobrinus, L. acidophilus, L. casei
among other microorganisms of the oral cavity.
Another study reports the high inhibitory capacity
of isopanduratin A against S. mutans, S. sobrinus,
S. sanguinis and S. salivarius 8.
Stevia rebaudiana (Bertoni), one of the 407 species
of the genus Stevia and one of the only two whose
leaves contain a sweetening substance, has been
used by native people as a sweetener and for medicinal purposes. It is a shrub originally from Paraguay and Brazil, which occasionally grows wild9. It
was described botanically in 1905 by naturalist
Moisés Santiago Bertoni, as an herbaceous plant 40
to 80 cm tall9 of the family Compositae10. In addition to being a non-caloric sweetener known in
many parts of the world, it has hypoglycemiant,
antioxidant and antihypertensive action11-14. Another advantage is that no toxic or genotoxic activity
has been found in the complete extracts obtained
from Stevia rebaudiana Bertoni leaves15.
There are studies reporting antimicrobial activity of
extracts obtained from Stevia rebaudiana on fungi
and Gram-positive and Gram-negative bacteria11,16,17.
None of these studies includes an evaluation of Stevia
rebaudiana extracts on the microorganisms involved
in dental caries. The aim of this study was to evaluate
the antibacterial activity of extracts in hexane, methanol, ethanol, ethyl acetate and chloroform from Stevia rebaudiana Bertoni leaves against bacteria that
are important in dental caries and oral health.
MATERIALS AND METHODS
Bacteria
The following 16 bacterial strains were used to evaluate the antimicrobial activity of the extracts obtained from Stevia rebaudina: Streptococcus mutans
Acta Odontol. Latinoam. 2012
ATCC 25175, Streptococcus mutans ATCC 31989,
Streptococcus mitis 804 NCTC 3165, Streptococcus salivarius NCTC 8606, Lactobacillus acidophilus ATCC 4365, Streptococcus rattus FA 1 (G),
Streptococcus mutans C67-1, Streptococcus cricetus AHT, Streptococcus mutans Ingbritt, Lactobacillus plantarum 748, Lactobacillus casei 475,
Lactobacillus brevis, Streptococcus mutans 35FS3,
Streptococcus mutans 35FS1, Streptococcus mutans
29FS2 and Streptococcus sobrinus CIO 428. In
order to reconstitute and confirm their viability for
the assays, the lyophilized strains were re-suspended in brain heart infusion (BHI) broth and incubated at 37 ºC for 48 hours in anaerobic conditions
(H2:CO2:N2; 10:10:80) so that they would grow
well, after which they were transferred to BHI agar
for isolating.
Extracts
Dry Stevia rebaudiana leaves (Agricultura Colombiana) were powdered in a mill until 800 grams
were collected. Extracts were obtained in ethanol,
methanol, ethyl acetate, chloroform and hexane
using the cold soaking technique. Sixty grams of
powdered Stevia rebaudiana leaves were soaked in
250 ml of each solvent and placed in a mechanical
shaker (Precision Reciprocal Shaking Bath, Precision Scientific; USA) at 150 rpm and 37ºC for 24
hours. Then they were filtered through filter paper
(Spezialpapier Filtrak, GMBH; Germany) to remove any leaf residue. The extracts were immediately
filtered again through 0.45 µm filters (Filter Bottle
Top, Sigma Chemical Company; USA) to remove
bacteria and ensure that they were free from contamination. Then they were concentrated at low pressure in a rotary evaporator (Buchi B-169,
Vacuum-System; Germany) until they were dry. A
microbiological test was performed on each concentrated extract to ensure that there was no bacterial contamination at the end of the process. Finally,
the concentrated extracts were used to prepare 4
concentrations (15 mg/ml, 30 mg/ml, 60 mg/ml and
120 mg/ml).
Microbiological Assays
The antimicrobial activity of the extracts on bacteria was tested using the well diffusion method described by Dobner et al.18. A pure culture of each
bacterium to be tested was used to prepare a suspension in trypticase soy broth and adjusted by tur-
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Antibacterial activity of Stevia rebaudiana
173
bidimetry to 0.5 on the Mac Farland scale. From
this suspension, 100 µl were taken and added to 20
ml of Mueller Hinton agar (liquid and sterile),
mixed and poured into Petri dishes. The agar was
allowed to solidify, and after waiting 20 - 30 minutes, a sterile Pasteur pipette was used to make 4 to 5
0.5 cm wells in the agar in each dish. Thirty µl of
each of the 5 extracts was placed in the wells (this
volume fits exactly into the wells without overflowing onto the surface of the culture medium). Vancomycin 180 µg/ml and Azithromycin 150 µg/ml
were used as positive controls and each of the solvents as negative controls. The dishes were incubated at 37 ºC for 24-72 hours under anaerobic
conditions (H2:CO2:N2; 10:10:80).
After the incubation, the presence or absence of
inhibition zones was determined. Their diameter
was measured and the minimal inhibitory concentrations (MIC: lowest concentration of the extract
that produces an inhibition zone of at least 6 mm)
determined. Each assay was performed in triplicate
and the average reported in mm.
RESULTS
Final yield (dry weight -expressed as a percentagethat was finally obtained from processing 60 grams
of S. rebaudiana leaves) for each extract in etha-
nol, methanol, ethyl acetate, chloroform and hexane was, respectively, 22%, 16%, 10%, 14 % and
0.9%. None of the 5 negative controls had antimicrobial activity. The positive controls (Vancomycin 180µg/ml and Azithromycin 150 µg/ml) had
variable inhibitory activity on the 16 strains included in the study, with values ranging from 18 mm
to 25 mm.
The MIC of the extracts in hexane, methanol,
ethanol, ethyl acetate and chloroform on the 16
bacterial strains of the genera Streptococcus and
Lactobacillus were, respectively 30 mg/ml, 120
mg/ml, 120 mg/ml, 60 mg/ml and 60 mg/ml.
Table 1 shows the results obtained with extracts in
hexane, methanol, ethanol, ethyl acetate and chloroform on the 16 microorganisms studied. The
inhibition zones of the MIC for the 5 extracts on
the bacterial strains were variable, ranging from 9
mm to 17.3 mm. The hexane extract, which had the
lowest MIC, produced the following inhibition
zones, in mm: 10.0, 11.0, 12.0, 10.0, 9.3, 10.6,
10.3, 10.6, 10.3, 9.6, 12.6, 9.6, 13.6, 14.3, 14.0 and
13.3. The inhibition zones of the 5 extracts were
slightly higher for the 4 Lactobacillus strains than
for the 12 Streptococcus strains, as the lowest value
for inhibition zone was 12.3 mm and the highest
was 17.3 mm.
Table 1: Antibacterial activity of the Stevia rebaudiana Bertoni leaf extracts against the 16 bacterial strains.
Bacteria
S. mutans ATCC 25175
S. mutans ATCC 31989
S. mutans C67-1
S. mutans Ingbritt
S. mutans 35FS3
S. mutans 35FS1
S. mutans 29FS2
S. sobrinus CIO 428
S. mitis 804 NCTC 3165
S. salivrius NCTC 8606
S. rattus FA1(G)
S. cricetus AHT
L. acidophilus ATCC 4365
L. plantarum 748
L. casei 475
L. brevis
Hexane
30 mg/ml
Methanol
120 mg/ml
Extracts
Ethanol
120 mg/ml
Ethyl acetate
60 mg/ml
Chloroform
60 mg/ml
10.0
11.0
12.0
10.0
9.3
10.6
10.3
10.6
10.3
9.6
12.6
9.6
13.6
14.3
14.0
13.3
9.3
9.0
9.3
9.0
9.0
9.0
10.0
9.3
8.6
9.0
13.0
9.0
13.0
12.3
13.0
13.0
9.0
10.3
10.0
9.3
9.3
9.3
11.0
10.3
9.3
10.0
13.0
9.6
14.0
13.3
14.0
13.6
10.0
10.3
10.6
11.0
11.3
10.3
10.6
11.0
9.0
9.3
12.6
10.3
15.0
15.3
15.3
14.6
12.0
11.6
12.3
12.3
10.3
11.0
9.3
10.6
10.3
11.6
13.3
11.3
17.3
16.0
14.3
14.6
The values of the inhibition zones at the minimal inhibitory concentration are expressed in mm.
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F. Gamboa, M. Chaves
DISCUSSION
New substances with pharmacological potential
have been searched for and applied since ancient
times. During the last twenty years, there has been
a revival of interest in research into natural products, as they make up 50% of the drugs used clinically in developed countries, of which 25% come
from higher plants5.
This is the first study to determine the antimicrobial
activity of 5 extracts obtained from Stevia rebaudiana leaves on 16 Gram-positive bacterial species of
the genera Streptococcus and Lactobacillus, which
are important for dental caries and oral health. Existing research to date has only proved the antimicrobial activity of extracts of Stevia rebaudiana leaves
on fungi, rotavirus virus and very different bacteria
from those evaluated in this study11,16,17,19-23.
The results of this study match those of Tadhani et
al.16, Ghosh et al.17 and Taware et al.19 regarding the
activity of extracts in hexane, methanol, ethanol,
ethyl acetate and chloroform on Gram-positive and
Gram-negative bacterial species. In the study by Tadhani16, the antimicrobial activity of the extracts in
water, methanol, ethyl acetate and hexane is highly
variable and broad against Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus, Serratia marcescens, Pseudomonas aeruginosa, Bacillus
megaterium, Escherichia coli, Proteus vulgaris, Rhizopus oligosporus and Aspergillus niger. The extract
in water is only active against B. subtilis and S.
aureus, and the other three extracts are highly active
against all the microorganisms evaluated. The extract
in hexane has the greatest activity, including the
Gram-positive (B. subtilis, S. aureus, M. luteus) with
inhibition zones ranging from 8.67 to 35.3 mm. The
study by Ghosh17 reports the antimicrobial potential
of extracts in 6 solvents against 4 fungi and 6 bacterial species: Alternaria solani, Helminthosporium
solani, A. niger, Penicillium chrysogenum, E. coli, B.
subtilis, Enterococcus faecalis, Proteus mirabilis, P.
aeruginosa and S. aureus; the Gram-positive species
(B. subtilis, Enterococcus faecalis y S. aureus) were
susceptible to all 6 extracts (petroleum ether, cyclo-
hexane, chloroform, water, acetone and ethanol) at a
concentration of 250 µg/ml using the plate dilution
method. The study by Taware19 reports inhibitory
activity against S. aureus of extracts in ethyl acetate
obtained from S. rebaudiana callus.
Noticeable in the study by Abou-Arab et al.11 is the
lack of antimicrobial activity of the extracts in hexane, water, ethyl acetate and chloroform against three
Gram-positive microorganisms (Listeria monocytogenes, S. aureus and Bacillus cereus). In the study
by Jayaraman et al.21, the extracts in water and chloroform obtained form S. rebaudiana leaves have no
antimicrobial activity against S. aureus. The differences in the susceptibility of Gram-positive microorganisms to Stevia rebaudiana leaf extracts might
be due to particularities of the genus and species and
to differences in the organization of the cell wall1,2.
The results of this study show antimicrobial activity
of all the extracts at MICs between 30 mg/ml and
120 mg/ml. The ethanol and methanol extracts which
have the same MIC (120 mg/ml) have similar inhibitory activity. The inhibition zones for the hexane
extract are similar to those for ethanol and methanol,
nevertheless, the MIC (30 mg/ml) is lower. The inhibition zones for the 4 Lactobacillus species are
slightly higher in the ethyl acetate and chloroform
extracts (60 mg/ml), suggesting that they are the most
susceptible microorganisms. Their greater susceptibility to the different extracts may be due to their cell
wall structure or to the presence of a substance or
synergic mixture in the extracts that can penetrate the
bacteria easily and produce greater damage24,25.
Further studies are needed on the isolation, characterization and identification of substances present
in the extracts, and to determine the antibacterial
activity against a wide range of microorganisms that
are important in other oral infections. These substances could subsequently be used in toothpastes,
mouthwashes or other oral products with antibacterial potential. In addition, before any extracts or
substances obtained from S. rebaudiana leaves are
used therapeutically, it must be ensured that they
are not toxic to eukaryotic cells.
ACKNOWLEDGMENTS
This study was funded by the Dental Research Center (School
of Dentistry) of Pontificia Universidad Javeriana, Project
N° 3054.
CORRESPONDENCE
Dr. Fredy Gamboa
Departamento de Microbiología (Facultad de Ciencias) y Centro
de Investigaciones Odontológicas (Facultad de Odontología)
Pontificia Universidad Javeriana
Carrera 7 N° 40-62, Bogotá- Colombia
E-mail: [email protected]
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Antibacterial activity of Stevia rebaudiana
175
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